Multifactor transcriptional control of alternative oxidase induction integrates diverse environmental inputs to enable fungal virulence

Metabolic flexibility enables fungi to invade challenging host environments. In Candida albicans, a common cause of life-threatening infections in humans, an important contributor to flexibility is alternative oxidase (Aox) activity. Dramatic induction of this activity occurs under respiratory-stress conditions, which impair the classical electron transport chain (ETC). Here, we show that deletion of the inducible AOX2 gene cripples C. albicans virulence in mice by increasing immune recognition. To investigate further, we examined transcriptional regulation of AOX2 in molecular detail under host-relevant, ETC-inhibitory conditions. We found that multiple transcription factors, including Rtg1/Rtg3, Cwt1/Zcf11, and Zcf2, bind and regulate the AOX2 promoter, conferring thousand-fold levels of inducibility to AOX2 in response to distinct environmental stressors. Further dissection of this complex promoter revealed how integration of stimuli ranging from reactive species of oxygen, nitrogen, and sulfur to reduced copper availability is achieved at the transcriptional level to regulate AOX2 induction and enable pathogenesis.

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Policy information about availability of computer code Data collection TECAN infinite M NANO+/F200 Pro/SPARK plate readers were used for OD600 and luminescence measurements. Bio-Rad CFX384 was used for qPCR.

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Bio-Rad CFX Manager was used to export qPCR data. GraphPad Prism 7 was used to plot RT-qPCR data (bar graphs, line graphs and heat maps), screening data, and survival curves. This software was used to calculate all statistical significance for these assays as described. Java TreeView 1.1.6r4 was used to plot heat maps for growth data. Magellan 7.2 was used to collect data from TECAN plate readers. ZEN pro was used to capture and process fluorescence microscopic images.
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March 2021
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Investigators were not blinded when assessing the anti-CD45 IHC data. Three noncontiguous kidney sections from three independent mice were evaluated by multiple (three) investigators independently and the same conclusion was reached. All uncropped images are provided in a supplementary figure. Investigators were not blinded for classical molecular biology and biochemistry experiments as this was not necessary due to the use of statistical analysis to generate conclusions.

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Experiments were performed with 8-to 10-week-old female BALB/c mice (n=8 per experimental group) from Charles River Laboratories (Strain code 028). Animals were provided with fresh water and chow ad libitum. Housing was performed under a 12:12 light: dark cycle in a well-ventilated room (10-15 air changes per hour) with stabilized temperature (68-79 degrees Fahrenheit) and humidity (30-70%).

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Female mice were used in the study given their overall smaller size, which has been optimized for the C. albicans inoculum used to establish both systemic and commensal infection. Historically, female mice have been used for C. albicans injections as well due to the fact that female mice are easier to handle and do not fight when caged together, enabling adequate optimization of the inoculum dose (PMID: 35366989).
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